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| United States Patent |
5,585,255
|
|
Tsukada
, et al.
|
December 17, 1996
|
Bile acid sulfate sulfatase gene, plasmid containing said gene and
method of producing bile acid sulfate sulfatase
Abstract
The present invention relates to a bile acid sulfate sulfatase gene coding
for the amino acid sequence as shown in FIG. 4 and the gene derived from
bacteria which belongs to genus Pseudomonas testosteroni, or a bile acid
sulfate sulfatase gene as shown in FIG. 3, a plasmid containing the gene,
a method for producing a bile acid sulfate sulfatase, and a bile acid
sulfate sulfatase.
| Inventors:
|
Tsukada; Yoji (Kyoto, JP);
Tazuke; Yasuhiko (Ashiya, JP);
Okada; Shigenori (Uji, JP);
Adachi; Kenichi (Uji, JP)
|
| Assignee:
|
Marukin Shoyu Co., Ltd. (JP)
|
| Appl. No.:
|
140104 |
| Filed:
|
October 26, 1993 |
| PCT Filed:
|
February 26, 1993
|
| PCT NO:
|
PCT/JP93/00244
|
| 371 Date:
|
October 26, 1993
|
| 102(e) Date:
|
October 26, 1993
|
| PCT PUB.NO.:
|
WO93/17114 |
| PCT PUB. Date:
|
September 2, 1993 |
Foreign Application Priority Data
| Current U.S. Class: |
435/196; 435/320.1; 536/23.2 |
| Intern'l Class: |
C12N 009/16; C12N 015/55; C12N 015/63 |
| Field of Search: |
536/23.7,23.2
435/320.1,240.2,252.3,196
|
References Cited
U.S. Patent Documents
| 5091305 | Feb., 1992 | Sugimori et al. | 435/19.
|
| 5100795 | Mar., 1992 | Sugimori et al. | 435/196.
|
Other References
Hunkapiller et al., Meth. Enzymol. 91:227-236 (1983).
Lathe, J. Mol. Biol. 183:1-12 (1985).
Mashige et al., Clin. Chem. 27:1352-1356 (1981).
|
Primary Examiner: Wax; Robert A.
Assistant Examiner: Grimes; Eric
Attorney, Agent or Firm: Knobbe Martens Olson & Bear
Claims
We claim:
1. An isolated polynucleotide sequence coding for a bile acid sulfate
sulfatase, which has the polynucleotide sequence of SEQ ID NO:1.
2. An isolated polynucleotide sequence coding for a bile acid sulfate
sulfatase, which has the polynucleotide sequence of SEQ ID NO:3.
3. An isolated polynucleotide sequence coding for a bile acid sulfate
sulfatase, which has the polynucleotide sequence of SEQ ID NO:4.
4. A plasmid containing the polynucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3 and 4.
5. A method for producing bile acid sulfate sulfatase which comprises the
steps of:
a. extracting chromosomal DNA from a Pseudomonas testosteroni strain having
bile acid sulfate sulfatase activity;
b. digesting said chromosomal DNA with the restriction enzymes Pstl and
Smal to obtain at least a DNA fragment having a size of 2.36 kb;
c. inserting said DNA fragment into a vector DNA to obtain a recombinant
DNA;
d. transforming an Escherichia coli strain with said recombinant DNA;
e. screening a transformant harboring said recombinant DNA containing said
DNA fragment of 2.36 kb by assaying the bile acid sulfate sulfatase
activity;
f. cultivating said transformant in a culture medium to produce bile acid
sulfate sulfatase; and
g. recovering bile acid sulfate sulfatase from the culture.
6. The method for producing bile acid sulfate sulfatase according to claim
5, wherein step (b) comprises the steps of:
i. digesting said chromosomal DNA with the restriction enzyme EcoRI so as
to recover DNA fragments having a size of 3 kb to 7 kb;
ii. inserting said DNA fragments into vector DNAs to obtain a recombinant
DNA;
iii. transforming an Escherichia coli strain with said recombinant DNA
obtained to obtain a transformant producing bile acid sulfate sulfatase;
and
iv. subcloning said DNA fragment in said recombinant DNA contained in said
transformant by cleaving the DNA fragment with the restriction enzymes
Pstl and Smal, followed by inserting the cleaved fragment into a vector
DNA to obtain a recombinant DNA.
7. The method for producing bile acid sulfate sulfatase according to claim
5, wherein said DNA fragment in step (b) comprises a polynucleotide coding
for a peptide having the amino acid sequence of SEQ ID NO:2.
8. The method for producing bile acid sulfate sulfatase according to claim
7, wherein said DNA fragment comprises a polynucleotide having the
sequence selected from the group consisting of SEQ ID NOs: 1, 3 and 4.
9. The method for producing bile acid sulfate sulfatase according to claim
5, wherein said vector DNA in step (c) is the plasmid pUC18.
10. The method for producing bile acid sulfate sulfatase according to claim
5, wherein said Escherichia coli strain in step (d) is Escherichia coli
JM109.
11. The method for producing bile acid sulfate sulfatase according to claim
5, wherein said screened transformant in step (e) is a transformed
Escherichia coli with the accession number FERM BP-3715.
Description
TECHNICAL FIELD
The present invention relates to bile acid sulfate sulfatase and a gene
therefor, a plasmid containing the gene coding for said protein, a
transformant capable of producing bile acid sulfate sulfatase, and a
method of producing bile acid sulfate sulfatase.
BACKGROUND ART
The present inventor previously searched for an enzyme capable of
efficiently hydrolyze 3.alpha.-sulfuric acid esters of sulfated bile acids
for the purpose of enabling enzymatic assay of sulfated bile acids in
blood or urine. As a result, it was found that the bacterial species
Pseudomonas testosteroni, which belongs to the genus Pseuomonas, produces
the desired enzyme, bile acid sulfate sulfatase. The bile acid sulfate
sulfatase produced by said bacterial species is characterized in that it
acts on 3.alpha.-sulfated bile acids, leading to the formation of
3.beta.-hydroxy bile acids. Therefore, it was made possible to assay
3.alpha.-sulfated bile acids by oxidizing said 3.beta.-hydroxy bile acids
to 3-oxobile acids under the action of .beta.-hydroxysteroid dehydrogenase
in the presence of .beta.-NAD, which is a coenzyme for said dehydrogenase,
with simultaneous reduction of .beta.-NAD to NADH, and assaying the
thus-formed NADH by a per se known method (cf. Japanese Unexamined Patent
Publication No. 02-145,183).
However, such a method of producing bile acid sulfate sulfatase as
mentioned above has drawbacks. Thus, it is an indispensable condition that
cholic acid or the like, which is expensive, should be added, as an
inducer substrate, to the medium. Moreover, the yield of bile acid sulfate
sulfatase is low and the production procedure is rather complicated.
Accordingly, it is an object of the present invention to provide a method
of producing 3.alpha.-bile acid sulfate sulfatase in high yields and at a
low cost in an easy and simple manner.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a restriction enzyme map of a recombinant plasmid named
pABS106.
FIG. 2 shows a restriction enzyme map of a recombinant plasmid named
pABS101.
FIG. 3 shows the complete base sequence of the bile acid sulfate sulfatase
gene (SEQ ID NO:1).
FIG. 4 shows the amino acid sequence of the peptide obtained by translation
of the bile acid sulfate sulfatase gene (SEQ ID NO:2).
DISCLOSURE OF THE INVENTION
The present inventor found that when a transformant (e.g. Escherichia coli)
obtained by introduction of a recombinant DNA constructed by inserting,
into a vector DNA (e.g. plasmid vector), a 2.36 kb DNA fragment containing
the bile acid sulfate sulfatase gene region (1,509 bp) derived from a bile
acid sulfate sulfatase-producing bacterial strain belonging to Pseudomonas
testosteroni, for example Pseudomonas testosteroni ATCC 11996, is reared
and cultivated in an ordinary nutrient medium, said enzyme can be produced
efficiently in said transformant without adding any expensive substance
such as mentioned above to the medium. The present invention has been
completed based on this and other findings.
Thus, the present invention provides a bile acid sulfate sulfatase gene
derived from bacteria belonging to Pseudomonas testosteroni and containing
a DNA sequence coding for an amino acid sequence of the following formula
(A) (SEQ ID NO:2):
##STR1##
The invention also provides a bile acid sulfate sulfatase gene containing a
DNA sequence of the following formula (B) (SEQ ID NO:1):
##STR2##
The invention further provides a plasmid containing a DNA sequence coding
for the amino acid sequence of the above formula (A) as derived from
bacteria belonging to Pseudomonas testosteroni.
Still further, the invention provides bacteria belonging to Escherichia
coli and harboring a plasmid containing a DNA sequence coding for the
amino acid sequence shown by the above formula (A) as derived from
bacteria belonging to Pseudomonas testosteroni.
Furthermore, the invention provides a method of producing bile acid sulfate
sulfatase which comprises cultivating in a medium bacteria belonging to
the genus Escherichia that have acquired the ability to produce bile acid
sulfate sulfatase as a result of introduction thereinto of a novel
recombinant DNA constructed by insertion of a DNA fragment derived from a
bacterial strain belonging to Pseudomonas testosteroni and containing the
bile acid sulfate sulfatase gene defined by the restriction enzyme map
shown in FIG. 1 and recovering bile acid sulfate sulfatase from the
culture.
The invention further provides a method of producing bile acid sulfate
sulfatase as mentioned above in which the bile acid sulfate sulfatase gene
contains a DNA sequence coding for an amino acid sequence of the following
formula (A) (SEQ ID NO:2):
##STR3##
The invention still further provides bile acid sulfate sulfatase containing
an amino acid sequence of the following formula (A) (SEQ ID NO:2):
##STR4##
In the following, the invention is illustrated in more detail.
First, as the bacterial strain belonging to Pseudomonas testosteroni and to
serve as a bile acid sulfate sulfatase donor in the practice of the
invention, there may be mentioned, for example, Pseudomonas testosteroni
ATCC 11996 etc. Such bacterial strain is cultivated to give a culture. As
the method of cultivation, there may be mentioned, for example, the method
described in Japanese Unexamined Patent Publication No. 02-145,183 and the
like. Specifically, cultivation is conducted in a medium for general
bacteria, for example LB medium, generally under aerobic conditions, at
about 24.degree. to about 34.degree. C. for about 12 to about 24 hours.
From the culture obtained in that manner, cells are harvested by a
conventional method, for example by filtration, centrifugation, etc. The
chromosomal DNA can be obtained from the cells by the phenol method
[Biochimica et Biophysica Acta, vol. 72, pp. 619-629 (1963)], for
instance.
The thus-obtained chromosomal DNA is then inserted into a vector DNA. The
method of insertion is not limited to any particular one. Thus, for
example, the insertion can be effected by cleaving the chromosomal DNA and
vector DNA using a variety of restriction enzymes for preparing DNA
fragments and then mixing both DNA fragments together, followed by
treatment with a DNA ligase. As the restriction enzymes, there may be
mentioned, for example, BamHI, EcoRI, HindIII, PstI, SmaI, etc.
Then, the recombinant DNA obtained in the above manner is introduced into
host cells, in particular cells of a bacterial strain belonging to the
genus Escherichia, for example Escherichia coli JM109 or Escherichia coli
HB101, by a conventional method, for example by the calcium chloride
treatment method.
An Escherichia coli strain harboring the desired recombinant plasmid (with
the bile acid sulfate sulfatase inserted therein) can be selected and
isolated by allowing the disrupted cell extract to act on a sulfated bile
acids, for example cholic acid 3-sulfate, and detecting the desulfation
product isocholic acid (5.beta.-cholanic
acid-3.beta.,7.alpha.,12.alpha.-triol) by thin layer chromatography (TLC).
Then, the recombinant plasmid with a bile acid sulfate sulfatase
gene-containing DNA fragment inserted therein is extracted and purified
from the strain obtained in the above manner and showing bile acid sulfate
sulfatase activity by using the method described in Molecular Cloning,
Second Edition, vol. 1, pages 21-24, for instance. Said recombinant
plasmid is further subjected to subcloning according to a conventional
procedure. In this manner, a novel recombinant plasmid with a 2.36 kb DNA
fragment, which contains the bile acid sulfate sulfatase gene, inserted
therein is obtained (cf. FIG. 1).
Using the above-mentioned bile acid sulfate sulfatase gene-containing
recombinant plasmid, the complete base sequence of the bile acid sulfate
sulfatase gene alone is analyzed (cf. FIG. 3) and, then, the amino acid
sequence of the polypeptide actually obtained by translation of the gene
having said base sequence is ascertained (cf. FIG. 4). As shown later in
an example, the polypeptide obtainable is not the peptide resulting from
faithful translation of FIG. 3, but a peptide beginning with His as
resulting from deletion of 28 or more amino acid residues on the
N-terminal side. This is the finding obtained for the first time in
connection with the present invention.
For producing bile acid sulfate sulfatase using the Escherichia coli strain
obtained as mentioned above and harboring the recombinant plasmid with the
bile acid sulfate sulfatase gene-containing DNA fragment inserted therein,
said Escherichia coli strain is cultivated in the following manner to give
cultured cells.
Said Escherichia coli strain may be cultivated in the manner of ordinary
solid culture but is preferably cultivated by a liquid culture method. The
medium for cultivating said Escherichia coli strain may be any of those
synthetic, semisynthetic or natural media containing carbon sources,
nitrogen sources, inorganic compounds and other nutrients, and generally
used for bacterial culture. As the carbon sources utilizable in the above
media, there may be mentioned, for example, saccharide solutions
containing glucose, fructose, invert sugar, saccharified starch, sorbitol,
glycerol, etc., and organic acids such as pyruvic acid, malic acid,
succinic acid etc. As the nitrogen sources, there may be mentioned, for
example, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium
phosphate, ammonium hydroxide, ammonium tartrate, ammonium acetate, urea
and so forth. Peptone, yeast extract, meat extract, corn steep liquor and
the like can be used not only as carbon sources but also as nitrogen
sources. The inorganic compounds include monopotassium phosphate,
dipotassium phosphate, monosodium phosphate, disodium phosphate, magnesium
sulfate, magnesium chloride, potassium chloride, sodium chloride, ferrous
sulfate, ferrous chloride, ferric sulfate, ferric chloride, manganese
sulfate, manganese chloride, etc.
The cultivation period and cultivation temperature are not critical but are
preferably within the range of 25.degree. C. to 42.degree. C., preferably
around 30.degree. C., and 6 to 24 hours, preferably 8 to 14 hours,
respectively. Cultivation is carried out under these conditions in the
manner of ordinary shaking culture or submerged culture (cultivation with
aeration and agitation).
The enzyme in question is extracted from the cells obtained by the
cultivation. The extraction can be carried out by a conventional method
for extracting intracellular enzymes. Thus, for instance, the cells are
disrupted by ultrasonic treatment, any of various mechanical treatments,
or enzyme treatment, and the insoluble matter is removed by
centrifugation, for instance, whereby the enzyme can be recovered in the
supernatant. This crude enzyme can be purified by a combination of
appropriate techniques selected from among those generally known for
enzyme purification. Thus, for example, bile acid sulfate sulfatase can be
obtained by treatment for nucleic acid removal, salting out with ammonium
sulfate, ion exchange chromatography, gel filtration, etc.
In the gene of the present invention, the code for each amino acid may be
alternative, hence said gene may be any of those coding for the amino acid
sequence defined by formula (A). The amino acid sequence defined by
formula (A) is a minimum requirement. Thus, the gene defined by formula
(B) and the like genes longer than said sequence are also included within
the scope of the present invention. The DNA sequences beginning with any
of the three ATG codons numbered 1-3, (SEQ ID NO:1), 13-15 (SEQ ID NO:3)
and 22-24 (SEQ ID NO:4) in the DNA sequence defined by formula (B) are
also included within the scope of the present invention.
The gene of the invention, which encodes the amino acid sequence of formula
(A), remains within the scope of the invention even when a plurality of
amino acid residues on the N-terminal and/or C-terminal side are cleaved
off by means of an exopeptidase, for example carboxypeptidase, in the
range reserving the enzymatic activity.
The plasmid to be used as the vector DNA in the practice of the invention
preferably contains an appropriate selective marker and is preferably
amplifiable to a large copy number. As examples, there may be mentioned
pUC18, pUC19, pBR322, pTrc99A, etc.
The method of the invention consists in isolating a DNA fragment containing
the gene for bile acid sulfate sulfatase that Pseudomonas testosteroni
produces, constructing a recombinant plasmid containing said DNA fragment,
utilizing this to transform Escherichia coli, cultivating the transformant
to thereby cause intracellular production of a large quantity of bile acid
sulfate sulfatase, and recovering the same. The productivity of the
genetically engineered strain obtained in accordance with the invention is
about 100 times in the production of bile acid sulfate sulfatase as
compared with the original strain and thus facilitates the production of
the enzyme. The addition of an inducer substrate such as cholic acid,
which is expensive, for inducing bile acid sulfate sulfatase is no longer
necessary.
EXAMPLE
The following example is further illustrative of the present invention.
(1) Preparation of chromosomal DNA
Pseudomonas testosteroni ATCC 11996 having bile acid sulfate sulfatase
activity was inoculated into 750 ml of LB medium (10 g/L tryptone, 5 g/L
yeast extract, 10 g/L sodium chloride, pH 7.0), shaking culture was
conducted at 30.degree. C. for 12 hours and cells were then harvested by
centrifugation. Chromosomal DNA was extracted from the thus-obtained cells
by the phenol method [Biochimica et Biophysica Acta, vol. 72, pp. 619-629
(1963)]. About 5 mg of chromosomal DNA was thus obtained.
(2) Preparation of a recombinant plasmid by insertion of a chromosomal DNA
fragment
To 70 .mu.g of the chromosomal DNA obtained in (1) was added the
restriction enzyme EcoRI, and the DNA was partially digested by incubation
at 37.degree. C. for 30 minutes, 60 minutes or 90 minutes. The digests
thus obtained were combined and subjected to electrophoresis using a low
melting agarose gel, and DNA fragments with a size of 3 kb to 7 kb were
recovered. These were purified by treatment with phenol, treatment with
ether and precipitation with ethanol to give purified DNA fragments.
Separately, the restriction enzyme EcoRI was added to 2 .mu.g of the
plasmid pUC18 employed as the vector, and the plasmid was completely
cleaved by incubation at 37.degree. C. for 2 hours. The subsequent
purification by treatment with alkaline phosphatase, treatment with
phenol, treatment with ether and precipitation with ethanol gave
EcoRI-cleaved pUC18.
The thus-obtained plasmid pUC18 and the chromosomal DNA fragments mentioned
above were respectively dissolved in a buffer solution and the solutions
were combined. T4 DNA ligase was further added and the ligation reaction
was carried out at 10.degree. C. for 15 hours to give a recombinant
plasmid solution.
(3) Transformation of Escherichia coli with recombinant plasmids
Escherichia coli JM109 was shake-cultured in 50 ml of LB medium at
37.degree. C. for 2 hours, cells were collected by centrifugation and
suspended in 50 ml of 100 mM magnesium chloride solution, and the
suspension was maintained on ice for 5 minutes and then centrifuged. The
cells thus recovered were further suspended in 50 ml of 100 mM calcium
chloride solution, the suspension was maintained on ice for 1 hour and
then centrifuged. The cells thus recovered were again suspended in 4 ml of
100 mM calcium chloride solution. To 0.4 ml of this cell suspension was
added the recombinant plasmid solution prepared in (2). The mixture was
allowed to stand on ice for 30 minutes and then heat-treated at 42.degree.
C. for 90 seconds for incorporation of the plasmid DNA into cells of said
bacterial strain, i.e. for transformation. To the suspension of the
thus-transformed cells was added 4 ml of LB medium, and shaking culture
was performed at 37.degree. C. for 1 hour. The culture was then spread
onto an LB agar medium plate supplemented with 100 mg/L of ampicillin,
23.8 mg/L of IPTG (isopropyl 1-thio-.beta.-D-galactoside) and 20 mg/L of
X-Gal (5-bromo-4-chloro-3-indolyl .beta.-D-galactoside) and cultured at
37.degree. C. for 18 hours.
(4) Isolation of a transformant capable of producing bile acid sulfate
sulfatase.
A white colony formed on the agar plate in (3) was shake-cultured in 5 ml
of LB medium (containing 100 mg/L of ampicillin and 23.8 mg/L of IPTG) at
37.degree. C. for 13 hours and, then, cells were separated by
centrifugation. The cells were suspended in 0.5 ml of a buffer solution
and disrupted by sonication. The subsequent centrifugation gave a
bacterial cell extract. This cell extract was allowed to act on cholic
acid 3-sulfate, the substrate of bile acid sulfate sulfatase, at
30.degree. C. for at least 24 hours and isocholic acid, the reaction
product, was detected by thin layer chromatography. Thus was obtained a
transformant Escherichia coli strain capable of producing bile acid
sulfate sulfatase.
The recombinant plasmid contained in this transformant Escherichia coli was
named pABS101. A restriction enzyme map of said plasmid is shown in FIG.
2.
(5) Construction of a recombinant plasmid named pABS106
The DNA fragment inserted in pABS101 was subcloned. Thus, further cleavage
was effected using PstI and SmaI, fragments were inserted into the plasmid
pUC18, followed by introduction into Escherichia coli JM109 and further
subjected to bile acid sulfate sulfatase activity assay. The bile acid
sulfate sulfatase activity assay was performed by the method described in
Japanese Unexamined Patent Publication No. 02-145,183. Thus, 0.1 ml of 2.5
mM aqueous solution of lithochloic acid sulfate (Sigma), 0.2 ml of 15 mM
aqueous solution of .beta.-NAD (Oriental Yeast), 1.0 ml of 0.1 mM
Tris-hydrochloride buffer (pH 8.0) and 1.55 ml of distilled water were
placed in a quartz cell and, after equilibrated at 30.degree. C., 0.05 ml
of a solution of .beta.-hydroxysteroid dehydrogenase (Sigma) (10 U/ml) and
0.1 ml of each cell disruption product supernatant were added in that
order and the enzyme reaction was started at 30.degree. C. The increment
in absorbance at 340 nm in the early reaction stage was measured. As a
result, the existence of bile acid sulfate sulfatase activity was observed
in a 2.36 kb DNA fragment inserted in the plasmid pABS106 shown in FIG. 1.
The Escherichia coli strain harboring said plasmid introduced therein was
named Escherichia coli JM109/pABS106 and had been deposited with the
Fermentation Research Institute of the Agency of Industrial Science and
Technology under the designation Escherichia coli JM109/pABS106 and the
accession number FERM-3715.
(6) Production of bile acid sulfate sulfatase
Using Escherichia coli JM109 transformed by introduction of the bile acid
sulfate sulfatase gene-containing plasmid pABS106 mentioned above, bile
acid sulfate sulfatase was produced. One liter of a medium (containing 100
mg/L ampicillin; pH 6.0) comprising 2% yeast extract, 1% succinic acid and
0.01% manganese chloride was distributed, in 200-ml portions, into 2-liter
flasks for shaking culture, the above strain was inoculated into each
portion of the medium, and shaking culture was performed. About 8 hours
after initiation of the cultivation, IPTG was added to 0.1 mM, and shaking
culture was further continued for 4 hours. Cells were then harvested by
centrifugation and suspended in 30 mM phosphate buffer (pH 7.2). The cells
were disrupted using a sonic oscillator and the precipitate was removed by
centrifugation to give a crude enzyme solution. This crude enzyme solution
was deprived of nucleic acids using protamine sulfate and then subjected
to salting-out treatment using ammonium sulfate. The precipitate fractions
resulting from 33 to 70% saturation with ammonium sulfate were collected
and dialyzed against 10 mM phosphate buffer (pH 7.2). The dialyzate was
passed through a DEAE-cellulose (Whatman) column equilibrated with 10 mM
phosphate buffer (pH 7.2). The unadsorbed fraction obtained was
concentrated by salting out treatment with ammonium sulfate and then
passed through a DEAE-Sepharose (Pharmacia) column equilibrated with 5 mM
Tris-hydrochloride buffer (pH 8.0) containing 0.5 mM MnCl.sub.2. The
unadsorbed fraction obtained was concentrated by salting out treatment
with ammonium sulfate and thereafter subjected to gel filtration by
passing through a Sephacryl S-200 (Pharmacia) column equilibrated with 50
mM phosphate buffer (pH 6.8) containing 0.15 M NaCl and 0.5 mM MnCl.sub.2.
The active fraction was desalted by dialysis and obtained 500 units of
bile acid sulfate sulfatase.
The enzyme activity was measured by the above-mentioned method comprising
reacting the enzyme with lithocholic acid sulfate in the presence of
.beta.-NAD and .beta.-hydroxysteroid dehydrogenase at 30.degree. C. and
measuring the increment in absorbance at 340 nm in the early stage of
reaction. Each unit was defined as the quantity of enzyme forming 1
.mu.mol of NADH in one minute. Finally, the productivity of bile acid
sulfate sulfatase using the recombinant Escherichia coli JM109 became
about 100 times higher as compared with Pseudomonas testosteroni.
(7) Analysis of the base sequence of the bile acid sulfate sulfatase
gene-containing DNA
The base sequence of the recombinant plasmid pABS106 was analyzed by the
dideoxy method. For gel electrophoresis for the analysis, a 6%
polyacrylamide gel was used.
The thus-obtained complete base sequence of the bile acid sulfate sulfatase
gene portion alone is shown in FIG. 3, and the amino acid sequence of the
polypeptide obtained as a result of translation of said gene is shown in
FIG. 4.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 4
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1509 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: BILE ACID SULFATASE
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 106..1509
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATGAATGGAGCAATGGCAAACATGAGAAAAGTATCTCGCCTCTCCCGATACGCCTTTGCC60
ACAGCCCTGGCACTGAGCCAGTTCGGCACAGGCACGGCCAACGCCCACGATCAG114
HisAspGln
GATGATCGCGGTGGCTCGGGAGCGAAAAGCCCCGCTGTGCTTGCTGCC162
AspAspArgGlyGlySerGlyAlaLysSerProAlaValLeuAlaAla
51015
CGCGCCCAGGTGTTCAAGGCCAATCCGCAGATGGTCAGGTCCATCATG210
ArgAlaGlnValPheLysAlaAsnProGlnMetValArgSerIleMet
20253035
GAAGGCGGTGGCTTTGGCACCGAGCTGTCGTATGCAGTAGCCAACAGC258
GluGlyGlyGlyPheGlyThrGluLeuSerTyrAlaValAlaAsnSer
404550
ATGTACAGCCGAACCGACCAGAACGCCATTGCAGATGCCCGAGCCAAG306
MetTyrSerArgThrAspGlnAsnAlaIleAlaAspAlaArgAlaLys
556065
CTCAAAGTCGAGGCCGTGGCTCCACGCACCTGGCTGCTGCGTTTCCCC354
LeuLysValGluAlaValAlaProArgThrTrpLeuLeuArgPhePro
707580
ATCGTCAACGTGGTGGTCTTCGAGACCGACGAAGGCCTGGTCTTGGTC402
IleValAsnValValValPheGluThrAspGluGlyLeuValLeuVal
859095
GATAGCGGCTACGCACCTGCAGGCCCGGCCTTGGCCGAAACGCTGAAG450
AspSerGlyTyrAlaProAlaGlyProAlaLeuAlaGluThrLeuLys
100105110115
AAGCTCAGCAACAAGCCGTTGCACACCGTCATCCTCACGCACTTTCAT498
LysLeuSerAsnLysProLeuHisThrValIleLeuThrHisPheHis
120125130
GCCGACCATGCCTTTGGCGCCTGGGCGTTGATGGACCAGAAGCCGCAT546
AlaAspHisAlaPheGlyAlaTrpAlaLeuMetAspGlnLysProHis
135140145
GTAGTGACCGAGCAGCGCTTCATCTCCCAGATGGAGCTGGACATGCGC594
ValValThrGluGlnArgPheIleSerGlnMetGluLeuAspMetArg
150155160
AGCAACGGTCTGATTGCACGCAACAACCAGCAAAGCGTGGCCGATGTG642
SerAsnGlyLeuIleAlaArgAsnAsnGlnGlnSerValAlaAspVal
165170175
CCCCGGACCTGGGCAGATGCAGTTCGGCCCACCCAGACCTTCAGGGAC690
ProArgThrTrpAlaAspAlaValArgProThrGlnThrPheArgAsp
180185190195
AAGACCACACTCAAAATTGGCGGCGAAGACTTTGTGCTGACCCATGCG738
LysThrThrLeuLysIleGlyGlyGluAspPheValLeuThrHisAla
200205210
CGCGGCGAGACCGAAGACCAGATATGGGTTGCCGTTCCAGGCCGGAAA786
ArgGlyGluThrGluAspGlnIleTrpValAlaValProGlyArgLys
215220225
ATCGTGGCCAGCGCGGATTATTTCCAGGGGTTTCTGCCCAATGCGGGC834
IleValAlaSerAlaAspTyrPheGlnGlyPheLeuProAsnAlaGly
230235240
AACGGCAAGCGCCGCCAGCGCTACCCCGAGGAGTGGGCCCGGGCCCTG882
AsnGlyLysArgArgGlnArgTyrProGluGluTrpAlaArgAlaLeu
245250255
CGCGACATGGCAGCACTCAAACCCGAGCTGCTGCTGCCGGCGCATGGT930
ArgAspMetAlaAlaLeuLysProGluLeuLeuLeuProAlaHisGly
260265270275
CCGGCCATCACCAAGCCCGAGGAAATTCAGGACCGACTGCCCGCCCAG978
ProAlaIleThrLysProGluGluIleGlnAspArgLeuProAlaGln
280285290
GCCCAGATGCTGGACAGCATCTCCAGGCAAGTGGTGGCCGGCCTGAAC1026
AlaGlnMetLeuAspSerIleSerArgGlnValValAlaGlyLeuAsn
295300305
AGCGGAGTACGCCGCGATCAGGTCATTGAAAAAGTCGCACTGCCGCCG1074
SerGlyValArgArgAspGlnValIleGluLysValAlaLeuProPro
310315320
GAGCTGGCCCGGCGAAGCGATGCACGCGAGCTATATGTGTCTGCCAAA1122
GluLeuAlaArgArgSerAspAlaArgGluLeuTyrValSerAlaLys
325330335
GACATAGGCCGCATGGTGGTCAGCGAGTACAGCGGCTGGTGGGACGAT1170
AspIleGlyArgMetValValSerGluTyrSerGlyTrpTrpAspAsp
340345350355
ATTCCATCGCACTGGCGCCCGGCGTCCCTGGCCAATGAGGCCAAAGAA1218
IleProSerHisTrpArgProAlaSerLeuAlaAsnGluAlaLysGlu
360365370
ATCGTGCAGCTAGCTGGCGGTGCCAGGCCGGTGATTCAGCGTGCAGTG1266
IleValGlnLeuAlaGlyGlyAlaArgProValIleGlnArgAlaVal
375380385
GCGCTGGCAGACAGCAATCCGGAGCTGGCCTCCCATCTGGCCGACTGG1314
AlaLeuAlaAspSerAsnProGluLeuAlaSerHisLeuAlaAspTrp
390395400
GCCTGGTATGCAGACAGCGATGACACCGAGGTGGCTCAAGGCGCACTG1362
AlaTrpTyrAlaAspSerAspAspThrGluValAlaGlnGlyAlaLeu
405410415
AAGGTCTATTCCGCGCGTGTTGCCAAGCCTCTGCCCACGCAGGAAGTG1410
LysValTyrSerAlaArgValAlaLysProLeuProThrGlnGluVal
420425430435
CTGGTCTATGCCGAGCACATGGTGCGCCTGCAGCTCAAGCTCAATGAG1458
LeuValTyrAlaGluHisMetValArgLeuGlnLeuLysLeuAsnGlu
440445450
CTGAACAGCACACGCGCGGCCAGCGCCAGTCAGAGCAGCAAAGCGCAT1506
LeuAsnSerThrArgAlaAlaSerAlaSerGlnSerSerLysAlaHis
455460465
TAA1509
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 467 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
HisAspGlnAspAspArgGlyGlySerGlyAlaLysSerProAlaVal
151015
LeuAlaAlaArgAlaGlnValPheLysAlaAsnProGlnMetValArg
202530
SerIleMetGluGlyGlyGlyPheGlyThrGluLeuSerTyrAlaVal
354045
AlaAsnSerMetTyrSerArgThrAspGlnAsnAlaIleAlaAspAla
505560
ArgAlaLysLeuLysValGluAlaValAlaProArgThrTrpLeuLeu
65707580
ArgPheProIleValAsnValValValPheGluThrAspGluGlyLeu
859095
ValLeuValAspSerGlyTyrAlaProAlaGlyProAlaLeuAlaGlu
100105110
ThrLeuLysLysLeuSerAsnLysProLeuHisThrValIleLeuThr
115120125
HisPheHisAlaAspHisAlaPheGlyAlaTrpAlaLeuMetAspGln
130135140
LysProHisValValThrGluGlnArgPheIleSerGlnMetGluLeu
145150155160
AspMetArgSerAsnGlyLeuIleAlaArgAsnAsnGlnGlnSerVal
165170175
AlaAspValProArgThrTrpAlaAspAlaValArgProThrGlnThr
180185190
PheArgAspLysThrThrLeuLysIleGlyGlyGluAspPheValLeu
195200205
ThrHisAlaArgGlyGluThrGluAspGlnIleTrpValAlaValPro
210215220
GlyArgLysIleValAlaSerAlaAspTyrPheGlnGlyPheLeuPro
225230235240
AsnAlaGlyAsnGlyLysArgArgGlnArgTyrProGluGluTrpAla
245250255
ArgAlaLeuArgAspMetAlaAlaLeuLysProGluLeuLeuLeuPro
260265270
AlaHisGlyProAlaIleThrLysProGluGluIleGlnAspArgLeu
275280285
ProAlaGlnAlaGlnMetLeuAspSerIleSerArgGlnValValAla
290295300
GlyLeuAsnSerGlyValArgArgAspGlnValIleGluLysValAla
305310315320
LeuProProGluLeuAlaArgArgSerAspAlaArgGluLeuTyrVal
325330335
SerAlaLysAspIleGlyArgMetValValSerGluTyrSerGlyTrp
340345350
TrpAspAspIleProSerHisTrpArgProAlaSerLeuAlaAsnGlu
355360365
AlaLysGluIleValGlnLeuAlaGlyGlyAlaArgProValIleGln
370375380
ArgAlaValAlaLeuAlaAspSerAsnProGluLeuAlaSerHisLeu
385390395400
AlaAspTrpAlaTrpTyrAlaAspSerAspAspThrGluValAlaGln
405410415
GlyAlaLeuLysValTyrSerAlaArgValAlaLysProLeuProThr
420425430
GlnGluValLeuValTyrAlaGluHisMetValArgLeuGlnLeuLys
435440445
LeuAsnGluLeuAsnSerThrArgAlaAlaSerAlaSerGlnSerSer
450455460
LysAlaHis
465
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1497 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: BILE ACID SULFATASE
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 106..1509
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATGGCAAACATGAGAAAAGTATCTCGCCTCTCCCGATACGCCTTTGCCACAGCCCTGGCA60
CTGAGCCAGTTCGGCACAGGCACGGCCAACGCCCACGATCAGGATGATCGCGGTGGCTCG120
GGAGCGAAAAGCCCCGCTGTGCTTGCTGCCCGCGCCCAGGTGTTCAAGGCCAATCCGCAG180
ATGGTCAGGTCCATCATGGAAGGCGGTGGCTTTGGCACCGAGCTGTCGTATGCAGTAGCC240
AACAGCATGTACAGCCGAACCGACCAGAACGCCATTGCAGATGCCCGAGCCAAGCTCAAA300
GTCGAGGCCGTGGCTCCACGCACCTGGCTGCTGCGTTTCCCCATCGTCAACGTGGTGGTC360
TTCGAGACCGACGAAGGCCTGGTCTTGGTCGATAGCGGCTACGCACCTGCAGGCCCGGCC420
TTGGCCGAAACGCTGAAGAAGCTCAGCAACAAGCCGTTGCACACCGTCATCCTCACGCAC480
TTTCATGCCGACCATGCCTTTGGCGCCTGGGCGTTGATGGACCAGAAGCCGCATGTAGTG540
ACCGAGCAGCGCTTCATCTCCCAGATGGAGCTGGACATGCGCAGCAACGGTCTGATTGCA600
CGCAACAACCAGCAAAGCGTGGCCGATGTGCCCCGGACCTGGGCAGATGCAGTTCGGCCC660
ACCCAGACCTTCAGGGACAAGACCACACTCAAAATTGGCGGCGAAGACTTTGTGCTGACC720
CATGCGCGCGGCGAGACCGAAGACCAGATATGGGTTGCCGTTCCAGGCCGGAAAATCGTG780
GCCAGCGCGGATTATTTCCAGGGGTTTCTGCCCAATGCGGGCAACGGCAAGCGCCGCCAG840
CGCTACCCCGAGGAGTGGGCCCGGGCCCTGCGCGACATGGCAGCACTCAAACCCGAGCTG900
CTGCTGCCGGCGCATGGTCCGGCCATCACCAAGCCCGAGGAAATTCAGGACCGACTGCCC960
GCCCAGGCCCAGATGCTGGACAGCATCTCCAGGCAAGTGGTGGCCGGCCTGAACAGCGGA1020
GTACGCCGCGATCAGGTCATTGAAAAAGTCGCACTGCCGCCGGAGCTGGCCCGGCGAAGC1080
GATGCACGCGAGCTATATGTGTCTGCCAAAGACATAGGCCGCATGGTGGTCAGCGAGTAC1140
AGCGGCTGGTGGGACGATATTCCATCGCACTGGCGCCCGGCGTCCCTGGCCAATGAGGCC1200
AAAGAAATCGTGCAGCTAGCTGGCGGTGCCAGGCCGGTGATTCAGCGTGCAGTGGCGCTG1260
GCAGACAGCAATCCGGAGCTGGCCTCCCATCTGGCCGACTGGGCCTGGTATGCAGACAGC1320
GATGACACCGAGGTGGCTCAAGGCGCACTGAAGGTCTATTCCGCGCGTGTTGCCAAGCCT1380
CTGCCCACGCAGGAAGTGCTGGTCTATGCCGAGCACATGGTGCGCCTGCAGCTCAAGCTC1440
AATGAGCTGAACAGCACACGCGCGGCCAGCGCCAGTCAGAGCAGCAAAGCGCATTAA1497
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1488 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: BILE ACID SULFATASE
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 106..1509
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ATGAGAAAAGTATCTCGCCTCTCCCGATACGCCTTTGCCACAGCCCTGGCACTGAGCCAG60
TTCGGCACAGGCACGGCCAACGCCCACGATCAGGATGATCGCGGTGGCTCGGGAGCGAAA120
AGCCCCGCTGTGCTTGCTGCCCGCGCCCAGGTGTTCAAGGCCAATCCGCAGATGGTCAGG180
TCCATCATGGAAGGCGGTGGCTTTGGCACCGAGCTGTCGTATGCAGTAGCCAACAGCATG240
TACAGCCGAACCGACCAGAACGCCATTGCAGATGCCCGAGCCAAGCTCAAAGTCGAGGCC300
GTGGCTCCACGCACCTGGCTGCTGCGTTTCCCCATCGTCAACGTGGTGGTCTTCGAGACC360
GACGAAGGCCTGGTCTTGGTCGATAGCGGCTACGCACCTGCAGGCCCGGCCTTGGCCGAA420
ACGCTGAAGAAGCTCAGCAACAAGCCGTTGCACACCGTCATCCTCACGCACTTTCATGCC480
GACCATGCCTTTGGCGCCTGGGCGTTGATGGACCAGAAGCCGCATGTAGTGACCGAGCAG540
CGCTTCATCTCCCAGATGGAGCTGGACATGCGCAGCAACGGTCTGATTGCACGCAACAAC600
CAGCAAAGCGTGGCCGATGTGCCCCGGACCTGGGCAGATGCAGTTCGGCCCACCCAGACC660
TTCAGGGACAAGACCACACTCAAAATTGGCGGCGAAGACTTTGTGCTGACCCATGCGCGC720
GGCGAGACCGAAGACCAGATATGGGTTGCCGTTCCAGGCCGGAAAATCGTGGCCAGCGCG780
GATTATTTCCAGGGGTTTCTGCCCAATGCGGGCAACGGCAAGCGCCGCCAGCGCTACCCC840
GAGGAGTGGGCCCGGGCCCTGCGCGACATGGCAGCACTCAAACCCGAGCTGCTGCTGCCG900
GCGCATGGTCCGGCCATCACCAAGCCCGAGGAAATTCAGGACCGACTGCCCGCCCAGGCC960
CAGATGCTGGACAGCATCTCCAGGCAAGTGGTGGCCGGCCTGAACAGCGGAGTACGCCGC1020
GATCAGGTCATTGAAAAAGTCGCACTGCCGCCGGAGCTGGCCCGGCGAAGCGATGCACGC1080
GAGCTATATGTGTCTGCCAAAGACATAGGCCGCATGGTGGTCAGCGAGTACAGCGGCTGG1140
TGGGACGATATTCCATCGCACTGGCGCCCGGCGTCCCTGGCCAATGAGGCCAAAGAAATC1200
GTGCAGCTAGCTGGCGGTGCCAGGCCGGTGATTCAGCGTGCAGTGGCGCTGGCAGACAGC1260
AATCCGGAGCTGGCCTCCCATCTGGCCGACTGGGCCTGGTATGCAGACAGCGATGACACC1320
GAGGTGGCTCAAGGCGCACTGAAGGTCTATTCCGCGCGTGTTGCCAAGCCTCTGCCCACG1380
CAGGAAGTGCTGGTCTATGCCGAGCACATGGTGCGCCTGCAGCTCAAGCTCAATGAGCTG1440
AACAGCACACGCGCGGCCAGCGCCAGTCAGAGCAGCAAAGCGCATTAA1488
__________________________________________________________________________
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